Understanding the reaction mechanisms of enzymes is an important step towards designing inhibitors for therapeutic purposes and for biomimetic applications. Such reaction mechanisms involve kinetics that reveals the various steps of the reaction, the reaction rate constants and the associated activation energies. In addition, reaction mechanisms focus on identification of reaction intermediates and their structural transformations. Such transformations can be conformational changes of the enzyme, formation of enzyme-substrate complexes etc. A complete understanding of a reaction mechanism involves identification of reaction intermediates, evaluation of reaction kinetics and assessment of the sequence and rate of structural transformations. While the kinetic is usually studied by stop-flow techniques combined primarily with optical spectroscopic detection, the structural analysis often requires freeze quench approaches. In freeze quench approaches, snap shots of reaction intermediates are obtained by rapid freezing followed by spectroscopic analysis of the frozen sample. Spectroscopic analysis includes for example electron paramagnetic resonance (EPR) or extended X-ray absorption fine structure (EXAFS).
Rapid-freeze quench (RFQ)-EPR is an established method where two (or more) components are mixed at ambient temperature and after some delay the liquid is sprayed into a cold trap. The frozen particles are collected into an EPR tube for measurement. The standard time resolution of commercial RFQ apparatuses is currently in the ms range with a typical dead-times of about 5-10 ms. Shorter deadtimes (˜200 μs) can be obtained with home-built setups, such as the tangential mixer set up. The deadtime is the shortest reaction time that can be accessed with the RFQ device.
RFQ-EPR is currently applied mostly to biological systems, specifically enzymatic reactions and the samples are usually analyzed by X-band (9.5 GHz) continuous wave (CW) EPR. Trapped samples were also subjected to interrogation by high resolution EPR techniques. One example is electron nuclear double resonance (ENDOR) which provides ligand hyperfine couplings that are essential for further characterization of the trapped intermediates. Recently, distance measurements by double electron-electron resonance (DEER) were also applied to freeze-quenched samples to follow protein folding.
High resolution X-band EPR techniques are usually less sensitive than CW EPR and therefore require large amount of sample for a complete set of measurements (e.g. 7-10 samples of ˜50 μl, 0.1-1 mM, each). This causes a difficulty that prevents the routine combination of RFQ with such high resolution EPR techniques. One way to overcome this obstacle is by coupling RFQ with sensitive high field EPR spectrometers. For example, the sample volume for W-band (95 GHz) EPR in systems employing a cavity (e.g. Bruker commercial spectrometers) is ˜2 μl with a concentration range comparable to that used for X-band. This is a ˜20 fold reduction in sample amount compared to X-band measurements. This difference becomes most significant when a set of 7-10 samples is required for a complete RFQ experiment. Another advantage of high field EPR and ENDOR is their increased spectral resolution. Currently, efficient high field RFQ-EPR is unavailable, primarily because of the difficulty to handle small sample tubes (capillaries) and the lack of an apparatus offering high yield and efficient collection of small volume samples.
The first application of RFQ high-field EPR was reported by Schunemann et. al. where the reaction of cytochrome P450cam with peroxy acids revealed the formation of tyrosyl radicals as intermediates. The freeze quenched samples were subjected to CW EPR measurements at 95, 195 and 285 GHz. The mixing and freezing were done using a commercial system from Update Instruments. The collection system for W-band was modified to be suitable for working with fragile quartz capillaries. In this approach, although high resolution is obtained by the high field measurements, a large amount of protein is required and most of it is wasted. In another study, RFQ with conventional and high-field EPR was utilized to resolve a unique heme and radical intermediates in the reaction of M. tuberculosis KatG with hydrogen peroxide. The mixing was done by a commercial system (Update instruments) and the liquid was sprayed onto a set of two rotating copper wheels partially immersed in liquid nitrogen. A home-built platform immersed into liquid nitrogen was used for sample collection.
An effective RFQ apparatus for W- or D-band (140 MHz) EPR spectrometers that uses a cavity should be able to use microfluidic technology to take full advantage of the small sample volume needed. Such a set-up has been introduced by Lin et. al. for X-band application where the primary objective was to shorten the dead-time. A microfluidic mixer was used and the freezing was achieved by spraying the sample onto cold copper-beryllium rotating wheels. Another microfluidic RFQ set up for high field EPR with a modified design for a single sample collection on vertical copper rotating wheels has been recently reported by J. Manzerova et. al. in J. Mag. Res. 213 (2011) 32-45. The drawback of the sample collection used in this setup is that after each time point the rotating wheels have to be cleaned, and the dead-time reported was rather long (˜30 ms). Further, the total amount of sample needed for a series of samples collected at different reaction times is not evident.
RFQ-EPR suffers from the difficulties of producing and manipulating small samples and from the inefficiency of sample freezing and sample collection processes in experiments that involve series of samples.